1. Field of the Invention
Treponema hyodysenteriae (T. hyo) is an anaerobic spirochete that causes swine dysentery [D. J. Taylor et al., Br. Vet. J. 127: lviii-lxi (1971); D. L. Harris et al., Vet. Med./Small Anim. Clin. 67: 61-64 (1972)], a severe, mucohemorrhagic intestinal disease of economic importance to the U.S. pork industry [anonymous, "Research and Education Priorities," National Pork Producers Council, Des Moines, IA (1984)]. In addition, swine dysentery has been reported in every major pig-producing country [R. A. Roncalli et al., "Geographic Distribution of Swine Dysentery,"In Proceedings of the 4th International Congress of the Pig Veterinary Society, L. 17 (1976)].
Effective diagnosis of this disease is currently hindered by the time needed to culture T. hyo from fecal specimens (4-6 days), and the difficulty in distinguishing T. hyo from nonpathogenic spirochetes such as T. innocens and T. succinifaciens, that also inhabit the swine intestine. This invention relates to a diagnostic tool useful in a rapid and accurate assay for the presence of T. hyo in biological samples taken from swine.
2. Description of the Prior Art
Various methods have been used in the diagnosis of swine dysentery. These methods include examination of samples by microscopy and direct or indirect immunofluorescence [D. Hunter et al., Vet. Rec. 101: 303-304 (1977); C. N. Saunders et al., Vet. Rec. 94: 491-492 (1974)]. Serological methods include a passive hemolysis test [E. M. Jenkins et al., Inf. Imm. 14: 1106-1108 (1976)], microtitration agglutination test [L. A. Joens et al., J. Clin. Microbiol. 8: 293-298 (1978)], and ELISA [L. A. Joens et al., J. Clin. Microbiol. 15: 249-252 (1982); I. T. Egan et al., Am. J. Vet. Res. 44: 1323-1328 (1983)], which are all designed to detect an immune response against T. hyo in infected pigs.
The most widely used method for diagnosing swine dysentery is by streaking a fecal specimen on a blood agar plate containing the antibiotic spectinomycin [Songer et al., J. Clin. Microbiol. 4: 57-60 (1976)], and examining the plate for the presence of .beta.-hemolytic zones following 4-6 days incubation at 39.degree. C. under anaerobic conditions. However, the long incubation times required and a lack of sensitivity and specificity make this test inadequate.
Nucleic acid probes are a sensitive and rapid alternative to culture methods for the detection of pathogens [L. Palmer et al., "Selection of DNA Probes for Use in the Diagnosis of Infectious Disease," In Rapid Detection and Identification of Infectious Agents, D. T. Kingsbury and S. Falkow, eds., Academic Press, Orlando, FL, pages 211-218 (1985); F. C. Tenover, Clin. Microbiol. Rev. 1: 82-101 (1988)], particularly in the case of fastidious or noncultivatable organisms. DNA probes, for example, are now in common usage and can be custom designed in terms of their sizes and specificities for a variety of prospective applications. The sizes of these probes can range from entire plasmids (kilobases in size) down to simple 10- to 15-base synthesized oligonucleotides. DNA probes can be tailored to bind to different nucleic acids including DNA, ribosomal RNA (rRNA), and messenger RNA (mRNA). A given probe will bind to specific nucleotide sequences. A radioactive, enzymatic, or organic label bound to the probe allows it to be detected.
DNA probes to Campylobacter pylori [B. L. Wetherall et al., J. Med. Microbiol. 26: 257-263 (1988)], Mycobacteria sp. [P. D. Ellner et al., J. Clin. Microbiol. 26: 1349-1352 (1988)], and Pseudomonas fluorescens [H. Festl et al., Appl. Environ. Microbiol. 52: 1190-1194 (1986)] as well as other pathogens have been successfully developed.
DNA probes specific for rRNA sequences have been used successfully in the detection of Mycoplasma sp. [U. B. Gobel et al., J. Gen. Microbiol. 133: 1969-1974 (1987)], Proteus sp. [G. Haun et al., FEMS Microbiol. Lett. 43: 187-193 (1987)], and Bacterioides sp., Acinobacillus sp., and Haemophilus sp. [P. L. Chuba et al., J. Gen. Microbiol. 134: 1931-1938 (1988)]. No cross hybridization to closely related strains occurred and a detection limit of less than 5.times.10.sup.3 cells was found. Various diagnostic kits based on DNA probes are now available for use in clinical labs and undoubtedly more will follow.
DNA probes can also be used in the detection of sequence variation in the DNA of related organisms by restriction fragment length polymorphism (RFLP) analysis. When a sample of DNA is cut with a restriction endonuclease, the size of the DNA fragments generated can vary depending on the sequence variation in the DNA samples examined. Different restriction endonucleases enable the detection of different polymorphisms.
To analyze the variety of fragment sizes from different samples, the digested DNA is electrophoresed in an agarose gel to separate the fragments based on size. The gel is then stained with ethidium bromide and RFLPs are visually identified, or the fragments are transferred from the gel to a solid support and hybridized with a DNA probe which binds to the polymorphic fragment(s) of interest. The sensitivity and specificity of this technique depends on the restriction endonucleases and DNA probes used.
RFLP analysis is already being used in classifying bacteria [S. E. Douglas et al., Appl. Environ. Microbiol. 54: 3071-3078 (1988)] and amoebae [G. L. McLaughlin et al., J. Clin. Microbiol. 26: 1655-1658 (1988)], for agricultural genetics [J. S. Beckman, Bio/technology 6: 1061-1064 (1988)], and for DNA fingerprinting in human genetics [R. Schafer et al., Electrophoresis 9: 369-374 (1988)].